The blood-brain barrier (BBB) comprises the brain microvascular endothelial cells (BMECs) which line brain capillaries and control trafficking between the bloodstream and neural tissue. These properties are tightly regulated by the surrounding microenvironment (termed the neurovascular unit) throughout BBB development and into adulthood. While this barrier is essential for preserving healthy brain activity, its dysfunction and deregulation is implicated in a number of neurological diseases (Zlokovic, 2008). Moreover, an intact BBB serves as a major bottleneck for brain drug delivery (Pardridge, 2005). Unfortunately, studies involving BBB development and regulation can be difficult and time-consuming to conduct in vivo, and the ability to screen brain-penetrating therapeutics in vivo is restricted to a small number of researchers with technical expertise in such techniques. Thus, researchers often use more accessible platforms, i.e. in vitro BBB models, to study interactions between BMECs and the neurovascular unit and to conduct compound library screens for prospective BBB-permeant drugs.
In vitro BBB models are typically constructed using primary BMECs isolated from animal brain tissue, including bovine, porcine, rat, and mouse (reviewed extensively in (Deli, et al., 2005)). These BMECs are then co-cultured with combinations of cells of the neurovascular unit, such as neurons, pericytes, and/or astrocytes, to upregulate BBB properties (Nakagawa, et al., 2009; Nakagawa, et al., 2007; Weidenfeller, Svendsen, et al., 2007; Lippmann, et al. 2011). Models derived from animal tissue have proved extremely useful in studying various aspects of the BBB, such as developmental and regulatory mechanisms (Daneman, et al. 2009; Daneman, et al., 2010(a); Kuhnert, et al., 2010; Lee, et al., 2003; Wosik, et al., 2007), but it is generally well-accepted that owing to species differences, a robust human BBB model must be developed to screen therapeutics that can prospectively traverse the human BBB in vivo (Cecchelli, et al., 2007). Human BMEC sources for BBB models have previously included biopsied brain tissue (Bernas, et al., 2010); (Rubin, et al., 1991) and immortalized cell lines (Weksler, et al., 2005). Primary human BMECs typically possess moderate barrier properties but their availability and yield are both extremely low and thus this source of material cannot be scaled for large library screens. Immortalized BMECs exhibit prodigious growth from a clonal population but often have poor barrier properties and are thus not optimal for screening therapeutics. From a co-culture perspective, human neurons, astrocytes, and pericytes can also be difficult to obtain from primary tissue sources in large enough quantities for modeling purposes. These collective issues have hindered the creation of a robust and readily accessible human BBB in vitro model for several decades (Deli, et al., 2005).
Applicants' previous work has demonstrated that stem cells may be attractive candidates to replace primary cells in human BBB models. Applicants have shown that human neural progenitor cells (hNPCs) may be differentiated to a defined mixture of neurons and astrocytes capable of inducing BBB properties in rat BMECs (Lippmann, et al., 2011). Further, Applicants recently demonstrated that human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), could be differentiated into endothelial cells possessing BBB properties (Lippmann, et al., 2012).
Needed in the art are fully-human BBB models, modulator-enhanced BBB models, BBB models under optimized media conditions, and BBB models having high absolute values of transendothelial electrical resistance TEER (e.g., >5000 Ω×cm2).